Dna barcoding-based method for rapid identification of lycium chinensis

ABSTRACT

Disclosed in the present invention is a trnL-trnF barcode and a DNA barcoding-based method for rapid identification of Lycium chinensis, relating to the technical field of identification of Lycium chinensis varieties. A Lycium chinensis phylogenetic tree is constructed based on the present DNA barcodes, and used in the study of the intraspecific and interspecific phylogeny of Lycium chinensis. The present invention further provides trnL-trnF barcode database of Lycium chinensis samples. By performing sequence alignment of trnL-trnF sequence of the sample to be identified and the trnL-trnF barcode database of Lycium chinensis samples, the Lycium chinensis varieties can be effectively identified and their interspecific relationship can be determined, providing an effective basis for the classification and identification of Lycium chinensis varieties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(Sequence-List-NXGQUSN-202008061.txt; Size: 53,000 bytes; and Date ofCreation: Mar. 14, 2021) is herein incorporated by reference in itsentirety.

CROSS REFERENCE TO RELATED APPLICATION

The present application is based upon and claims priority to ChineseApplication No. 2020103478862, filed on Apr. 28, 2020, and entitled“method for rapid identification of Lycium Chinensis based on DNAbarcode”, the entire contents of which are incorporated herein byreference

REFERENCE Technical Field

The present invention generally relates to the technical field ofvariety identification for Lycium chinensis, and specifically relates toa method for rapid identification of Lycium Chinensis based on DNAbarcode.

Background Art

Lycium Chinensis (Wolfberry) is rich in LBP (lycium barbarumpolysaccharide), betaine, carotenoids, and a variety of unsaturatedfatty acids, etc. It has the functions of anti-oxidation, anti-tumor,delaying aging, strengthening immunity, softening blood vessels andlowering blood lipid, etc. It is an important medicinal and edible plantresource in China.

Compared with breeding of other crops, the breeding of Lycium chinensishas the following shortcomings and drawbacks: firstly, there arerelatively few practical production varieties, only four new varieties(Ningqi-1, Ningqi-4, Ningqi-5 and Ningqi-7) are widely used in theproduction; in addition, they have relatively single uses and cannotadapt to the diversified development of Lycium chinensis industry;secondly, the breeding methods are relatively very few, with longbreeding cycle. In recent years, with the rapid development of molecularmarker technology, it can provide more effective judgment basis forin-depth understanding of plant gene polymorphism, targeted selection ofparents, and early identification of hybrid offspring, etc., therebyimproving the breeding efficiency. Through population selection, hybridbreeding, and cell fusion technologies, diversified breeding theoreticalsystem and technical system can be established and new multi-purposevarieties of Lycium chinensis can be cultivated, which is of greatsignificance to the improvement of the research level and sustainabledevelopment of Lycium chinensis industry.

DNA barcoding (DNA barcode) can be used to recognize and identify targetvarieties using one or a few DNA fragments. It is characterized bysimple operation, high accuracy, and rapid identification, etc.Presently, it has become a new research area and hotspot of interest inmodern biological taxonomy. In recent years, researchers at home andabroad have carried out active exploration and studies on DNA barcodegene sequences suitable for plant identification.

Chinese patent application CN110229927A titled “a DNA barcoding-basedmethod for identifying Heiguo Lycium chinensis and uses thereof”provides a method for identifying Heiguo Lycium chinensis based on DNAbarcoding. The DNA barcode gene sequence for identifying Heiguo Lyciumchinensis is LRITS2 (the second internal transcribed spacer of ribosomalRNA)/LRpsbA-trnH (a non-coding region between the chloroplast genes psbAand trnH). The DNA barcode sequence LRITS2/LRpsbA-trnH for identifyingHeiguo Lycium chinensis can be used simultaneously or one of them can beselected. The invention can identify Heiguo Lycium chinensis rawmaterials efficiently and accurately, to prevent similar confusing orcounterfeit products, in addition, it can be used for the identificationof fruit powder, fruit shreds, etc. It has important application valueand great social benefits to guarantee food safety and consumer rightsand interests.

The article titled “Identification of Lycium Germplasm Resources Basedon the matK Barcode Sequence” discloses the identification and analysisof 10 test materials of Lycium germplasm resources using the matK geneas the barcode coding sequence through DNA barcode technology, so as toobtain the theoretical basis of identifying Lycium plants at themolecular level. According to the method, sequence alignment isperformed using the ClustalX software, the sequence information isobtained by Mega7.0 and the difference between sequences is compared,finally a phylogenetic tree is constructed based on the K2P model. ThematK sequence has a total length of 936 bp, with 933 conserved sites and3 variable sites. The average GC content is 33.3%, and the basetransition transversion value is 1.8. Its phylogenetic tree is dividedinto two branches. Heiguo, Huangguobian and Changji Lycium chinensis areclustered into a branch, and other varieties are clustered into abranch, and each branch has a high Bootstrap value.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method for rapididentification of Lycium chinensis based on DNA barcode. The inventorsof this present application found, there exist problems of uncleargenetic background of Lycium germplasm resources and lagging inexcavation and utilization of excellent resources for the varietyidentification of Lycium chinensis. The present invention provides a DNAbarcode—the trnL-trnF barcode and a method based on it for rapididentification of Lycium chinensis. The present invention provides achloroplast-based spacer sequence, and also provides a rapid molecularmarker identification method of Lycium chinensis with representativegermplasm sources such as Heiguo Lycium chinensis, Huangguo Lyciumchinensis, Yuanguo Lycium chinensis, Hongzhi Lycium chinensis, localvarieties of Lycium barbarum, local varieties of Beifang, Xinjiang,Yunnan, Hebei, as well as hybrid populations, space-mutated populationsand ploidy populations, etc, which can be used for the identification ofLycium chinensis varieties.

The present invention provides a method for identifying Lycium chinensisvarieties and determining interspecific relationship based on DNAbarcoding. In addition, a trnL-trnF barcode database is provided, whichcan be used to effectively identify the Lycium chinensis varieties anddetermine the interspecific relationship of Lycium chinensis, providingeffective basis for Lycium chinensis varieties.

The present invention provides a trnL-trnF barcode and a DNAbarcoding-based method for rapid identification of Lycium chinensis.

The identifiable Lycium chinensis varieties include Ningqi-1 (L.barbarum Linn), Ningqi-2, Ningqi-3, Ningqi-4, Ningqi-5, Ningqi-6,Ningqi-7, Ningnongqi-9, Huangguobian Lycium chinensis (L. barbarum Linn.var. auranticarpum K. F. Ching var. nov.), Heiguo Lycium chinensis(Lycium ruthenicum Murr.), Ningnongqi-5 (L. barbarum Linn), BeifangLycium chinensis (Lycium chinense MilL. var. potaninii (Pojark.) A. M.Lu), Damaye Lycium chinensis (Damaye (L. barbarum Linn), Baihua Lyciumchinensis (Baihua (L. barbarum)) Zhongguo Lycium chinensis (L. ChinenseMill. var.), Yunnan Lycium chinensis (Lycium yunnanense Kuang et A. M.Lu), Mansheng Lycium chinensis (Manshenggouqi (L. barbarum)), ZibingLycium chinensis (Ziguogouqi (L. barbarum)), Hongzhi Lycium chinensis(Lycium dasystemum), Xiaomaye Lycium chinensis (Xiaomaye (L. barbarumLinn)), Xinjiang Lycium chinensis (Lycium dasystemum Pojark), Mengqi-1,Ningqicai-1, black hybrid space-mutated Lycium chinensis, space-mutatedLycium chinensis, Yuanguo Lycium chinensis, Lycium chinensis-9001,Ninggxia Huangguo Lycium chinensis, Changji Lycium chinensis, HebeiLycium chinensis, etc.

Preferably, the DNA barcoding-based method for rapid identification ofLycium chinensis, comprising the following steps:

1) extracting genomic DNA from Lycium chinensis samples;

2) amplifying trnL-trnF barcode sequence fragments using the extractedgenomic DNA as a template and the primers with nucleotide sequencesshown in SEQ ID NO. 35 and SEQ ID NO. 36 to obtain a PCR product;

3) sequencing the PCR product; and

4) constructing a phylogenetic tree and identifying Lycium chinensis.

Further, in step 1), the genomic DNA is extracted using a kit.

Preferably, a DNA secure Plant Kit is used to extract genomic DNA in thestep 1).

Further, the DNA extraction using the kit includes the following steps:

(1A) Extraction of DNA

The fresh and tender leaves of Lycium chinensis samples to be tested aretaken as samples, and put into a 5 ml cryotube and marked, then put theminto liquid nitrogen immediately, and stored at −80° C. The total DNA isextracted using a new plant genomic DNA extraction kit (DNA secure PlantKit).

The extraction method is as follows:

i) Taking 100 g sample and grinding in a multifunctional high-efficiencybiological sample preparation apparatus with a speed of 22 times/s for 2minutes. Immediately adding 400 ul of buffer LP1 and 6 ul RNase A (10mg/ml), oscillating for 1 min and placing at room temperature for 10min.

ii) Adding 130 ul of buffer LP2, mix well, and oscillating for 1 min.

iii) Centrifuging at 12000 rpm for 5 min, and transferring thesupernatant to a new centrifuge tube.

iv) Adding 1.5 times the volume of buffer LP3 (make sure that absoluteethanol has been added before use), shaking immediately and mixingthoroughly for 15 sec. At this time, flocculent precipitation may occur.

v) Adding the solution and flocculent precipitate obtained in theprevious step into an adsorption column CB3 (the adsorption column isput into the collection tube), centrifuging at 12000 rpm for 30 s,discarding the waste liquid, and putting the adsorption column CB3 intothe collection tube.

vi) Adding 600 ul of rinse solution PW to the adsorption column CB3(check if absolute ethanol has been added before use), centrifuging at12000 rpm for 30 s, discarding the waste liquid, and then putting theadsorption column CB3 into the collection tube. (Note: If the adsorptioncolumn membrane is green, add 500 ul of absolute ethanol to theadsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discardthe waste liquid, and put the adsorption column CB3 into the collectiontube).

vii) Repeating the step vi).

viii) Putting the adsorption column CB3 back into the collection tube,centrifuging at 12000 rpm for 2 minutes, and discarding the wasteliquid; placing the adsorption column CB3 at room temperature for 15 minto thoroughly remove the remaining rinse solution in the adsorptionmaterial.

ix) Transferring the adsorption column CB3 to a clean centrifuge tube,and adding 100 ul of elution buffer TE into the middle of the adsorptionmembrane, leaving it at room temperature for 2 minutes, centrifuging at12000 rpm for 2 minutes, and collecting the solution into the centrifugetube.

x) Repeating step ix). The DNA product is stored at −80° C. to preventDNA degradation.

(1B) Detection of DNA Concentration and Purity

i) Detection by Agarose Gel Electrophoresis

1.2% agarose gel is prepared with 1.2 g agarose and 100 ml 1*TAE buffer.4 ul ddH2O+1 ul DNA sample (undiluted)+1 ul 6*loading buffer are addedto a PCR tube to perform agarose gel electrophoresis, and the testresults are observed under a UV gel imaging system.

ii) Detection by UV Spectrophotometer

The UV spectrophotometer is preheated in advance, and 99 ul ddH2O+1 ulDNA sample (undiluted) are added to the PCR tube for detection. The testresults show the sample concentration and the ratio of OD₂₆₀/OD₂₈₀, andthe value of OD₂₆₀/OD₂₈₀ should be 1.7-1.9. If ddH2O instead of elutionbuffer is used for elution, the ratio will be lower because the pH valueand the presence of ions will affect the light absorption value, but itdoes not indicate low purity.

Preferably, in the step 2), the PCR amplification reaction system is: i)pre-denaturizing at 94 reaction sys ii) denaturizing at 94t 94 reactionsystem is: 55° C. for 30 s (the annealing temperature can be adjustedwithin the range of 58-60° C.), extending at 72° C. for 2 min, 35cycles; iii) keeping warm at 72° C. for 10 min; and iv) storing at 4°C.; performing detection of PCR product by 1.0% agarose gelelectrophoresis, and observing the amplification results under a UV gelimaging system.

Further, in the DNA barcoding-based method for rapid identification ofthe present invention, step 3) is sequencing the PCR product obtained instep 2). The sequencing method in the step 3) is as follows:

(3A) PCR Product Cloning:

Recovering target band(s) with AxyPrep DNA gel recovery kit, anddetecting by 1.2% agarose gel electrophoresis. The purified target DNAis used as a sequencing template. The recovered product is ligated tothe T vector (pGEM-T) using pLB zero background rapid cloning kit, thentransferred to E. coli DH5a for culture. The blue-white spot screeningmethod is used to screen positive colonies and PCR detection of coloniesis carried out. The amplification results are observed under a UV gelimaging system.

(3B) Sequencing and Analysis:

Sequencing the DNA sequence of the colony of positive clone, andperforming homology sequences alignment with the published sequence inNCBI, to analyze the sequence. The operations are as follows:

In the present invention, after performing PCR detection on positivecolonies, the colonies containing target fragments (positive colony) arecultured in LB liquid medium, and 3 colonies are selected for eachmaterial and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencingby Sanger method, to obtain trnL-trnF sequence.

The obtained DNA barcode gene sequence is aligned with the publishedsequence in the NCBI database for homology. The Clustal X program isused to align the Lycium chinensis DNA barcode gene sequencerespectively. The base composition of the target sequence, the frequencyof base variation between sequences and the frequency of transition andtransversion between sequences and their ratios are calculated by thephylogenetic analysis software MEGA7.0, and a phylogenetic tree isconstructed to establish a trnL-trnF barcode database for identificationof varieties of Lycium chinensis.

Another object of the present invention is to provide a trnL-trnFbarcode database of Lycium chinensis samples constructed by the abovemethod, comprising 34 trnL-trnF barcodes, and the nucleotide sequencethereof is shown in SEQ ID NO. 1-34.

Another object of the present invention is to provide uses of thetrnL-trnF barcode database of Lycium chinensis samples in identifyingLycium chinensis varieties.

Preferably, uses of the trnL-trnF barcode database of Lycium chinensissamples in identifying Lycium chinensis varieties comprise the followingstep:

performing sequence alignment of trnL-trnF sequence of the sample to beidentified and the trnL-trnF barcode database of Lycium chinensissamples to identify the Lycium chinensis varieties.

Preferably, the method for obtaining the trnL-trnF sequence of thesample to be identified involves genomic DNA extraction, PCRamplification, and sequencing of PCR products, to obtain thecorresponding sequence, and the operation steps are the same as steps1), 2) and 3) in the DNA barcoding-based identification method of Lyciumchinensis varieties.

The present invention can achieve the following beneficial effects:

(1) A method for identification of Lycium chinensis varieties based ontrnL-trnF gene is established for the first time. It can be used toidentify Lycium barbarum, Huangguobian Lycium chinensis, Heiguo Lyciumchinensis, Beifang Lycium chinensis, Damaye Lycium chinensis, ZhongguoLycium chinensis, Yunnan Lycium chinensis, Mansheng Lycium chinensis,Zibing Lycium chinensis, Hongzhi Lycium chinensis, etc.

(2) The genetic diversity and genetic relationship of Lycium arerevealed based on trnL-trnF gene, to provide an effective basis for theidentification, classification and phylogenic study of Lycium chinensisvarieties.

(3) It can identify Lycium chinensis varieties accurately based ontrnL-trnF gene.

(4) The present invention further provides trnL-trnF barcode database ofLycium chinensis samples, covering Heiguo Lycium chinensis, HuangguoLycium chinensis, Yuanguo Lycium chinensis, Hongzhi Lycium chinensis,local varieties of Lycium barbarum, local varieties of Beifang,Xinjiang, Yunnan, Hebei, as well as hybrid populations, space mutationpopulations and ploidy populations, etc, all of them are representativegermplasms of Lycium chinensis nationwide; Therefore, it can provide aneffective basis for the classification and identification of Lyciumchinensis varieties.

By performing sequence alignment of trnL-trnF sequence of the sample tobe identified and the trnL-trnF barcode database of Lycium chinensissamples, the Lycium chinensis varieties can be effectively identifiedand their interspecific relationship can be determined. By identifyingthe interspecific relationship between Lycium chinensis to be tested andLycium chinensis in the barcode database, it provides an effective basisfor the classification and identification of Lycium chinensis varieties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA extraction and detection result of the Lyciumchinensis samples in Example 1 of the present invention, of which, laneM: marker (DL2000 DNA molecular marker); (a) DNA detection results oflanes 1 to 24 corresponding to sample numbers 1 to 24; (b) DNA detectionresults of lanes 25 to 34 corresponding to sample numbers 25 to 34.

FIG. 2 shows the PCR amplification result of trnL-trnF sequence of someLycium chinensis samples in Example 1 of the present invention, ofwhich, lane M: marker (DL2000 DNA molecular marker); lanes 1-2: PCRproducts of Mansheng Lycium chinensis; lanes 4-5: PCR products ofYuanguo Lycium chinensis; lanes 7-8: PCR products of Zibing. Lanes 1-2,4-5 and 7-8 show twice PCR results of different samples.

FIG. 3 shows the trnL-trnF sequence cloning result of Lycium chinensissample No. 32 in Example 1 of the present invention. Of which, M: marker(DL2000 DNA molecular marker), lane 1: negative clone; lanes 2 to 6:positive clones (results of multiple repeated tests).

FIG. 4 shows the NJ phylogenetic tree constructed from the trnL-trnFbarcodes in the trnL-trnF barcode database of Lycium chinensis samplesin Example 1 of the present invention.

FIG. 5 shows the NJ phylogenetic tree of Lycium chinensis samples to beidentified and part of the trnL-trnF barcodes in the database in Example1 of the present invention.

FIG. 6 shows the NJ phylogenetic tree of Lycium chinensis samples to beidentified and part of the trnL-trnF barcodes in the database in Example2 of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention is described below through specific embodiments.Unless otherwise specified, the technical means used in the presentinvention are all methods known to those skilled in the art. Inaddition, the embodiments should be understood as illustrative ratherthan limiting the scope of the invention, and the essence and scope ofthe present invention are only defined by the appended claims. For thoseskilled in the art, without departing from the essence and scope of thepresent invention, various changes or modifications to the materialcomponents and amount in these embodiments shall also fall into thescope of protection of the present invention. The present invention willbe further described below in conjunction with specific embodiments.

Example 1 Identification of Lycium chinensis Samples and Construction oftrnL-trnF Barcode Database of Lycium chinensis Samples

The invention will be further described in conjunction with specificembodiments.

1. Samples from trnL-trnF Barcode Database of Lycium chinensis Samples

In order to construct a trnL-trnF barcode database of Lycium chinensissamples, a total of 34 samples with partial similar morphology fromdifferent regions are collected, as shown in Table 1.

TABLE 1 Lycium plant samples (trnL-trnF barcode database of Lyciumchinensis samples) Germplasm Resource No. Latin name name Code type SEQID NO 1 Ningqi No. 1 (L. barbarum Ningqi-1 Ningqi1 Bred variety 1 Linn)2 Ningqi No. 2 (L. barbarum Ningqi-2 Ningqi2 Bred variety 2 Linn) 3Ningqi No. 3 (L. barbarum Ningqi-3 Ningqi3 Bred variety 3 Linn) 4 NingqiNo. 4 (L. barbarum Ningqi-4 Ningqi4 Bred variety 4 Linn) 5 Ningqi No. 5(L. barbarum Ningqi-5 Ningqi5 Bred variety 5 Linn) 6 Ningqi No. 6 (L.barbarum Ningqi-6 Ningqi6 Bred variety 6 Linn) 7 Ningqi No. 7 (L.barbarum Ningqi-7 Ningqi7 Bred variety 7 Linn) 8 L. barbarum LinnNingnongqi-9 Ningnongqi9 Bred variety 8 9 L. barbarum Linn. HuangguobianHuangguobian Bred variety 9 var. auranticarpum K. F. Ching var. nov. 10Mengqi 1(L. barbarum) Mengqi-1 Mengqi1 Bred variety 10 From printedpublication 5 11 Lycium ruthenicum Murr. Heiguo Heiguo Bred variety 11Lycium chinensis 12 From printed publication 2 Ningqicai-1 Ningqicai1Bred variety 12 13 L. barbarum Linn Ningnongqi-5 W-12-30 Space mutant 1314 From printed publication 1 HZ-13-01 HZ-13-01 Black hybrid 14 spacemutant 15 From printed publication 1 ZH-13-08 ZH-13-08 Space mutant 1516 From printed publication 1 W-12-27 W-12-27 Black hybrid 16 spacemutant 17 From printed publication 1 W-11-15 W-11-15 Black hybrid 17space mutant 18 From printed publication 1 W-13-26 W-13-26 Black hybrid18 space mutant 19 From printed publication 1 W-12-26 W-12-26 Blackhybrid 19 space mutant 20 Lycium chinenseMilL. var. Beifang BeifangIntroduced 20 potaninii (Pojark.) A. M. Lu Lycium variety chinensis 21From printed publication 1 Yuanguo Yuanguo Bred variety 21 Lyciumchinensis 22 From printed publication 4 9001 9001 Bred variety 22Ninggxia 23 From printed publication 3 Huangguo Huangguo Bred variety 2324 Damaye (L. barbarum Linn) Damaye Damaye Bred variety 24 25 Baihua(L.barbarum) Baihua Baihua Introduced 25 variety 26 L. chinenseMill. var.Zhongguo Zhongguo Introduced 26 Lycium variety chinensis 27 LyciumyunnanenseKuang et Yunnan Yunnan Introduced 27 A. M. Lu Lycium varietychinensis 28 Manshenggouqi (L. bararum) Mansheng Mansheng Introduced 28Lycium variety chinensis 29 Ziguogouqi (L. barbarum) Zibing ZibingIntroduced 29 variety 30 Lycium dasystemum Hongzhi Hongzhi Introduced 30variety 31 From printed publication 1 Hebei Hebei Introduced 31 Lyciumvariety chinensis 32 Xiaomaye (L. barbarum Linn) Xiaomaye Xiaomaye Bredvariety 32 33 From printed publication 1 Changji Changji Introduced 33Lycium variety chinensis 34 Lycium dasystemumPojark Xinjiang XinjiangIntroduced 34 Lycium variety chinensis Note: (Variety numbers 14-19, 21,31, and 33 are varieties disclosed in the printed publication 1: “WanRu, Wang Yajun, An Wei, et al. Identification of 21 Lycium plants basedon psbA-trnH sequence barcodes [J]. Jiangsu Agricultural Sciences, 2019,47(01): 64-67.”; variety number 12 is from Table 1 in the printedpublication 2: “a new method of identifying vegetable Lycium chinensis-nrDNA ITS sequencing method (English)[J]. Agricultural Science &Technology(2): 64-65 + 111”; Variety number 23 is from Table 1 in theprinted publication 3: “Shi Zhigang. Genetic diversity of 18 Ningxiawolfberry resources based on nrDNA ITS sequence [J]. Anhui AgriculturalSciences (24): 10379-10380”; variety number 22 is from Table 1 in thepublication 4: “Shi Zhigang. Genetic diversity of 18 Ningxia wolfberryresources based on nrDNA ITS sequence [J]. Anhui Agricultural Sciences(24): 10379-10380”; variety number 10 is from the publication 5: “YinYue, An Wei, Zhao Jianhua, et al. Transcriptome SSR information analysisand molecular marker development Heiguo Lycium chinensis [J]. Journal ofZhejiang Agriculture and Forestry University, 2019, 36(02): 215-221.”).2. Identification of Lycium chinensis samples and construction of thetrnL-trnF barcode database of Lycium chinensis samples

1) Extraction of DNA

In the Lycium chinensis germplasm resource nursery of Lycium EngineeringCenter, Ningxia Academy of Agricultural and Forestry Sciences, 34 partsof fresh and tender leaves of Lycium plants are taken as samples, andput into a 5 ml cryotube and marked, then put them into liquid nitrogenimmediately, and stored at −80° C. Sampling time: June 2018, samplinglocation: National Lycium Chinensis Germplasm Resource Bank in YinchuanCity, Ningxia.

The total DNA is extracted using a new plant genomic DNA extraction kit(DNA secure Plant Kit). The extraction method is as follows:

i) 100 g sample was taken and grinded in a multifunctionalhigh-efficiency biological sample preparation apparatus at 22 times/sfor 2 minutes. Immediately 400 ul of buffer LP1 and 6 ul RNase A (10mg/ml) was added. The mixture was oscillate for 1 min and placed at roomtemperature for 10 min.

ii) 130 ul of buffer LP2 was added, mixed well, and subjected tooscillate for 1 min. iii) Centrifugation at 12000 rpm for 5 min wasperformed. The supernatant was transferred to a new centrifuge tube.

iv) 1.5 times the volume of buffer LP3 (check if absolute ethanol hasbeen added before use) was added. The mixture was immediately shaken andmixed thoroughly for 15 s. At this time, flocculent precipitate mayoccur.

v) The solution and flocculent precipitate obtained in the previous stepiv) were added into an adsorption column CB3 (the adsorption column isput into the collection tube). After centrifugation at 12000 rpm for 30s, the waste liquid was discarded, and the adsorption column CB3 wasputted into the collection tube.

vi) 600 ul of rinse solution PW was added to the adsorption column CB3(check if absolute ethanol has been added before use). Aftercentrifugation at 12000 rpm for 30 s, the waste liquid was discarded,and the adsorption column CB3 was putted into the collection tube.(Note: If the adsorption column membrane was green, added 500 ul ofabsolute ethanol to the adsorption column CB3, centrifuged at 12000 rpmfor 30 seconds, discarded the waste liquid, and putted the adsorptioncolumn CB3 into the collection tube).

vii) Repeated the step vi).

viii) Putted the adsorption column CB3 back into the collection tube,centrifuged at 12000 rpm for 2 minutes, and discarded the waste liquid.The adsorption column CB3 was placed at room temperature for 15 min tothoroughly remove the remaining rinse solution in the adsorptionmaterial.

ix) Transferred the adsorption column CB3 to a clean centrifuge tube,and added 100 ul of elution buffer TE into the middle of the adsorptionmembrane, left it at room temperature for 2 minutes, centrifuged at12000 rpm for 2 minutes, and collected the solution into the centrifugetube.

x) Repeated step ix). The DNA product was stored at −80° C. to preventDNA degradation.

2) Detection of DNA Concentration and Purity

i) Detection by Agarose Gel Electrophoresis

1.2% agarose gel is prepared with 1.2 g agarose and 100 ml 1*TAE buffer.4 ul ddH2O+1 ul DNA sample (undiluted)+1 ul 6*loading buffer were addedto a PCR tube to perform agarose gel electrophoresis, and the testresults were observed under a UV gel imaging system, as shown in FIG. 1.

ii) Detection by UV Spectrophotometer

The UV spectrophotometer was preheated in advance, and 99 ul ddH2O+1 ulDNA sample (undiluted) were added to the PCR tube for detection. Thetest results showed the sample concentration and the ratio ofOD₂₆₀/OD₂₈₀, and the value of OD₂₆₀/OD₂₈₀ should be 1.7-1.9. If ddH2Oinstead of elution buffer was used for elution, the ratio will be lowerbecause the pH value and the presence of ions will affect the lightabsorption value, but it did not indicate low purity.

3) PCR Amplification

The DNA obtained in the step 1) was used as a template, and primers andother reagents required for amplification were added to perform PCRamplification. Refer to Table 2 and Table 3 for specific primers andamplification systems.

(1) Primer Design

The designed primer was as follows:

TABLE 2 Universal primers for DNA barcode gene trnL-trnF Primer SEQPrimer sequence name ID NO (5′ to 3′) trnL-trnF-F 35ATCGGTATCTAATGAATTCAATG trnL-trnF-R 36 CCCATACAAATTAATCATGTGCC

(2) PCR Reaction System

The genomic DNAs of the test material were amplified by PCR with theabove primers. The amplification system was shown in Table 3.

TABLE 3 DNA barcode reaction system Amplification system 50 ul systemPCR-Grade Water 15.0 ul 2X Ex taq Buffer (takara) 25.0 μl dNTP Mix (10mM) 1.0 μl Ex taq (takara) 1.0 μl DNA 5.0 μl primer F (10X) 1.5 μlprimer R (10X) 1.5 μlThe PCR reaction procedure: i) pre-denaturizing at 94° C. for 2 minutes;ii) denaturizing at 94° C. for 30 s, annealing at 55° C. for 30 s (theannealing temperature can be adjusted within the range of 58-60° C.),extending at 72° C. for 2 min, 35 cycles of denaturizing, annealing andextending; iii) preservation at 72° C. for 2 min; iv) storing at 4° C.Performed detection of PCR product by 1.0% agarose gel electrophoresis,and observed the amplification results under a UV gel imaging system.Taking the PCR products of Mansheng Lycium chinensis, Dahuangguo andZibing as examples, results are shown in FIG. 2. According to theposition of DNA Marker corresponding to the trnL-trnF sequence, thetotal length of the trnL-trnF sequence is about 1200 bp.

4) PCR Product Cloning:

The target band was recovered with AxyPrep DNA gel recovery kit, anddetected by 1.2% agarose gel electrophoresis. The purified target DNAwas used as a sequencing template. The recovered product was ligated tothe T vector (pGEM-T) using Lethal Based Simple Fast Cloning Kit, thentransferred to E. coli DH5a for culture. The blue-white spot screeningmethod was used to screen positive colonies and PCR detection ofcolonies was carried out. The amplification results were observed undera UV gel imaging system. Taken Xiaomaye Lycium chinensis (No. 32) as anexample, as shown in FIG. 3, the trnL-trnF gene had good amplificationresults, with clear bands and obvious cloning results.

5) Sequencing and Analysis:

The DNA sequencing of the screened bacteria liquid with positivecolonies was performed, and the homology sequences alignment wasperformed with the published sequence in NCBI (National Center forBiotechnology Information) database. The Clustal X program was used toalign the Lycium chinensis DNA barcode gene sequence, respectively. Theoperations were as follows:

after performing PCR detection on positive colonies, the coloniescontaining target fragments were cultured in LB liquid medium, and 3colonies were selected for each material and sent to Sangon Biotech(Shanghai) Co., Ltd. for sequencing, to obtain 34 parts of trnL-trnFsequence.

The obtained DNA barcode gene sequence was aligned with the publishedsequence in the NCBI database for homology. The Clustal X program wasused to align the Lycium chinensis DNA barcode gene sequencerespectively, and the primer area was removed. The phylogenetic analysissoftware MEGA7.0 was used to conduct analysis, the obtained trnL-trnFsequence had a total length of 1156 bp, 1138 conservative sites,accounting for 98.4%, and 18 variation sites, accounting for 1.6%,including 9 information sites and 9 singleton sites. The base transitionand transversion value is 1.2 and the average GC content is 35.7%.

After sequence alignment analysis, for 12 samples (Huangguobian, HeiguoLycium chinensis, Ningnongqi-5, HZ-13-01, ZH-13-08, W-12-27, W-11-15,W-13-26, W-12-26, Zhongguo Lycium chinensis, Yunnan Lycium chinensis,Changji Lycium chinensis), a sequence segment with the length of 40 bpis inserted at 61 bp, namely, TGACATCACAACGAGATCCTAATCTCAAAACAAAAAGAAA,and a base A is deleted at 791 bp. Except for Zhongguo Lycium chinensisand Yunnan Lycium chinensis, base transitions happen at 38 bp, 44 bp, 49bp, 50 bp, 323 bp, 569 bp, and 731 bp, and base transversions happen at762 bp for the remaining 10 samples. In addition, 3 bases (TCT) areinserted at 45 bp for the 10 samples. Except for 4 samples ofHuangguobian, Zhongguo Lycium chinensis, Yunnan Lycium chinensis, andChangji Lycium chinensis, the other 8 samples have a 24 bp sequenceinserted at 487 bp, namely, GAATTGGTGTGAATCGATTCTACA. The basetransitions happen at 1025 bp for Heiguo Lycium chinensis, and basetransitions happen at 236 bp and 263 bp for Beifang Lycium chinensis.The base transitions happen at 253 bp and 1131 bp for Mansheng Lyciumchinensis, transition from T to C. Ningqi-4 and Yuananguo Lyciumchinensis have sequence deletion of 6 bp (AAGGAA) at 55 bp, and sequencedeletion of 10 bp (CCGACCCCCT) at 732 bp. Xinjiang Lycium chinensis hastransition from A to G at 605 bp. Ningqicai-1 has transversion of 3bases from TAT to ATA at 1151 bp.

Through sequence alignment and clustering analysis, the phylogenetictree is constructed as shown in FIG. 4. The clustering graph oftrnL-trnF barcode sequence is divided into two branches, Zhongguo Lyciumchinensis, Yunnan Lycium chinensis, space mutant of Heiguo hybrids,Huangguobian, Heiguo Lycium chinensis and Changji Lycium chinensis andChangji Lycium chinensis are clustered into one branch, among which,Zhongguo Lycium chinensis, Yunnan Lycium chinensis are separate groups,which have far genetic relationship with the other varieties. Theremaining 22 germplasms are clustered together, among which Ningqi-4 andYuanguo are clustered together, with the closest genetic relationship.According to the trnL-trnF sequence, 34 germplasm materials can bebasically identified (shown in FIG. 4), and each branch obtains abootstrap value greater than 50%. This result is consistent with theactual genetic relationship of 34 samples, indicating that trnL-trnFsequence can be used to identify Lycium chinensis varieties.

This proves that the DNA barcode provided by the present invention canbe used to construct a Lycium chinensis phylogenetic tree, and then usedin the study of the intraspecific and interspecific phylogeny of Lyciumchinensis. It further proves the effectiveness and feasibility of theDNA barcode provided by the present invention in identification,classification and phylogenetic study of Lycium chinensis varieties. Inaddition, in the embodiments of the present invention, trnL-trnF barcodedatabase is constructed based on the barcode trnL-trnF sequences. Thedatabase covers Heiguo Lycium chinensis, Huangguo Lycium chinensis,Yuanguo Lycium chinensis, Hongzhi Lycium chinensis, local varieties ofLycium barbarum, local varieties of Beifang, Xinjiang, Yunnan, Hebei, aswell as hybrid populations, space mutation populations and ploidypopulations, etc, all of them are representative germplasms of Lyciumchinensis nationwide; Therefore, it can provide an effective basis forthe classification and identification of Lycium chinensis varieties.

By performing sequence alignment of trnL-trnF sequence of the sample tobe identified and the trnL-trnF barcode database of Lycium chinensissamples, the Lycium chinensis varieties can be effectively identifiedand their interspecific relationship can be determined. By identifyingthe interspecific relationship between Lycium chinensis to be tested andLycium chinensis in the barcode database, it provides an effective basisfor the classification and identification of Lycium chinensis varieties.

Experimental Example 1 Identification of Lycium chinensis VarietiesUsing Barcode Database

1. Sampling

Three samples of Lycium chinensis to be tested (Nos. Tianjing-3,Zhutong, Baitiao) were selected, and sequence alignment was performedwith barcodes of part of Lycium chinensis samples trnL-trnF barcodedatabase in Example 1 for identification. The Lycium chinensis varietycannot be identified by morphological methods. In this experimentalexample, DNA barcode based method of the present invention was used foridentification.

TABLE 4 Number and origin of Lycium chinensis samples to be tested Typeof sample Origin Tianjing-3 Hebei Zhutong Ningxia Zhongning (Xinyang)Baitiao Ningxia

2. The procedures for DNA extraction and concentration detection, PCRamplification, PCR product cloning, sequence sequencing and analysis arethe same as those in Example 1.

3. Analysis of Sequence Results

The sequence alignment and clustering analysis were performed by MEGA7.0software. The phylogenetic tree was constructed using NJ method. Thecluster graph of trnL-trnF barcode sequence was divided into twobranches. The sample to be tested Baitiao and Yuananguo Lycium chinensis(barcode database, No. 21) were clustered together, with the closestgenetic relationship, indicating that Baitiao has a closer geneticrelationship with Yuananguo Lycium chinensis and Ningqi-4 (barcodedatabase, No. 4) in the 34 trnL-trnF barcode databases, and has closegenetic relationship with Ningqi-1 (barcode database, No. 1). Thesamples to be tested Zhutong and Tianjing-3 are separate groups. Zhutongand Ningqi-1 have close genetic relationship; Tianjing-3 andHuangguobian (barcode database, No. 9) and Heiguo (barcode database, No.11) have close genetic relationship. The Bootstrap value of each branchis greater than 60, which has a high credibility, indicating that thebarcode database constructed based on trnL-trnF barcode sequence and themethod of the present invention can be used to perform classificationand identification of varieties for samples from different regions.

The genetic distance calculation using MEGA7.0 and K2P model (Kimura2-parameter model) is shown in Table 5. The minimum genetic distancebetween Ningqi-1 and Yuananguo Lycium chinensis is 0.00093, and themaximum genetic distance between Baitiao and Heiguo Lycium chinensis is0.0110186.

TABLE 5 The genetic distance analysis of Lycium chinensis varietiesidentified by trnL-trnF Yuanguo Heiguo Lycium Lycium Ningqi-1 chinensisHuangguobian chinensis Tianjing-3 Zhutong Baitiao Ningqi-1 Yuanguo0.000933 Lycium chinensis Huangguobian 0.007390 0.008446 Heiguo 0.0083210.009392 0.000885 Lycium chinensis Tianjing-3 0.001842 0.002806 0.0089220.009822 Zhutong 0.001839 0.002802 0.009250 0.010183 0.003687 Baitiao0.001840 0.001867 0.009253 0.010186 0.003690 0.003684

Experimental Example 2 Identification of Heiguo Lycium chinensisVarieties Using Barcode Database

1. Sampling

Three samples of Lycium chinensis to be tested (Nos. B2, B3, H-13-08-05)are selected. The DNA barcode technology is used for identification inthis exoerimental example, and sequence alignment is performed withbarcodes of part of Lycium chinensis samples trnL-trnF barcode databasein Example 1. The Lycium chinensis varieties cannot be identified bymorphological methods.

TABLE 6 Number and origin of Lycium chinensis samples to be testedSample type Origin B2 Qinghai B3 Qinghai H-13-08-05 Ningxia

2. The procedures for DNA extraction and concentration detection, PCRamplification, PCR product cloning, sequence sequencing and analysis arethe same as those in Example 1.

3. Analysis of Sequence Results

The sequence alignment and clustering analysis are performed by MEGA7.0software. The phylogenetic tree is constructed using NJ method as shownin FIG. 6. The cluster graph of trnL-trnF barcode sequence is dividedinto two branches. The Hongguo Lycium chinensis and Heiguo Lyciumchinensis are clearly identified. Ningqi-1 and Zhongguo Lycium chinensisare clustered together, with the closest genetic relationship, belongingto Hongguo Lycium chinensis, and their bootstrap value with other sixHeiguo Lycium chinensis is 100, with high degree of credibility.

Among the 6 Heiguo Lycium chinensis samples, the test samples B2 andH-13-08-05 are clustered together, with the closest geneticrelationship, and their bootstrap value with Heiguo Lycium chinensis(barcode database, No. 11) is 60, with credibility. Heiguo Lyciumchinensis, W-12-27 (barcode database, No. 16), test sample B3 areseparate groups. The bootstrap value between the test sample B3 andW-12-27 is 63, with credibility, and the bootstrap value with HeiguoLycium chinensis is 39, indicating that Heiguo Lycium chinensis samplesthat cannot be morphologically identified in different regions can beclassified and identified based on the trnL-trnF barcode sequence andthe barcode database constructed by the method of the present invention,but the samples have high similarity, so it is only used as preliminaryidentification.

The genetic distance calculation using MEGA7.0 and K2P model (Kimura2-parameter model) is shown in Table 7. The minimum genetic distancebetween Ningqi-1 and Zhongguo Lycium chinensis is 0.00000, and themaximum genetic distance between Ningqi-1 and H-13-08-05 is 0.011122.

TABLE 7 Genetic distance analysis of Heiguo Lycium chinensis identifiedby trnL-trnF Zhongguo Heiguo Lycium Lycium Ningqi-1 chinensis chinensisW-12-27 B3 B2 H-13-08-05 Ningqi-1 Zhongguo 0.000000 Lycium chinensisHeiguo 0.008321 0.008024 Lycium chinensis W-12-27 0.007390 0.0071270.000885 B3 0.008321 0.008024 0.001771 0.000885 B2 0.009250 0.0089200.002658 0.001771 0.002658 H-13-08-05 0.011122 0.010724 0.0044410.003549 0.004441 0.005329

1. A DNA barcoding-based method for rapid identification of Lyciumchinensis, wherein the DNA barcoding is trnL-trnF barcoding.
 2. The DNAbarcoding-based method for rapid identification of Lycium chinensisaccording to claim 1, wherein, a trnL-trnF barcode database of Lyciumchinensis samples is constructed comprising 34 groups of trnL-trnFbarcode, the nucleotide sequence thereof is shown in SEQ ID NO. 1-34. 3.The DNA barcoding-based method for rapid identification of Lyciumchinensis according to claim 1, comprising the following steps: 1)extracting genomic DNA from Lycium chinensis samples; 2) amplifyingtrnL-trnF barcode sequence fragments using the genomic DNA as a templateand the primers with nucleotide sequence shown in SEQ ID NO. 35 and SEQID NO. 36 to obtain a PCR product; 3) sequencing the PCR product; and 4)constructing a phylogenetic tree and identifying Lycium chinensis. 4.The DNA barcoding-based method for rapid identification of Lyciumchinensis according to claim 3, wherein the genomic DNA is extractedusing a kit in the step 1).
 5. The DNA barcoding-based method for rapididentification of Lycium chinensis according to claim 3, wherein in thestep 2), the PCR amplification reaction system is: i) pre-denaturizingat 94° C. for 2 min; ii) denaturizing at 94° C. for 30 s, annealing at55° C. for 30 s, extending at 72° C. for 2 min, 35 cycles; iii)preservation at 72° C. for 2 min; and iv) storing at 4° C.; performingdetection of PCR product by 1.0% agarose gel electrophoresis, andobserving the amplification results under a UV gel imaging system. 6.The DNA barcoding-based method for rapid identification of Lyciumchinensis according to claim 3, wherein, a trnL-trnF barcode database ofLycium chinensis samples is constructed comprising 34 groups oftrnL-trnF barcode, the nucleotide sequence thereof is shown in SEQ IDNO. 1-34.
 7. The DNA barcoding-based method for rapid identification ofLycium chinensis according to claim 2, wherein, a trnL-trnF barcodedatabase of Lycium chinensis samples is constructed by the followings:analyzing the base composition of a DNA barcoding gene sequence;constructing the barcode database based on the parameters including thefrequency of base variation between sequences and the frequency oftransition and transversion between sequences and their ratios.
 8. TheDNA barcoding-based method for rapid identification of Lycium chinensisaccording to claim 2, wherein, obtaining the trnL-trnF sequence of thesample to be identified through genomic DNA extraction, PCRamplification and sequencing of PCR products; comparing sequencealignment of trnL-trnF sequence of the sample to be identified and thetrnL-trnF barcode database of Lycium chinensis samples.
 9. The DNAbarcoding-based method for rapid identification of Lycium chinensisaccording to claim 6, wherein, obtaining the trnL-trnF sequence of thesample to be identified through genomic DNA extraction, PCRamplification and sequencing of PCR products; comparing sequencealignment of trnL-trnF sequence of the sample to be identified and thetrnL-trnF barcode database of Lycium chinensis samples.